SUMMARY Barrier function is compromised in infectious and immune-mediated intestinal and systemic diseases. This program, now completing its fourth funding cycle, has been guided by our long-term goal of understanding intestinal epithelial barrier regulation at a fundamental, molecular level and defining how regulation and dysregulation impact disease. This knowledge is required for development of rational, mechanism-based therapeutic approaches. In previous cycles, we have made paradigm-shifting discoveries including recognition that continuous molecular remodeling occurs within the tight junction. This and other new insight provided by our previous work led us to explore the molecular interactions and functional consequences of protein interactions at tight junctions. Our preliminary data demonstrate novel activities of claudin-4, occludin, and ZO- 1 that are unrelated to their ability to form tight junctions. Using a structural approach, we have discovered that occludin tail phosphorylation masks the ZO-1 binding site, while dephosphorylation triggers conformational change that enhances binding to ZO-1. The resulting occludin/ZO-1 complexed then form stable interactions with claudin-2, which disrupt channel function. In vivo, we found that the severity of immune-mediated colitis was markedly reduced or increased in claudin-2 knockout or transgenic mice, respectively. We combined the in vitro structural and in vivo functional data to inhibit occludin phosphorylation, block claudin-2 channels, and attenuate immune-mediated colitis in vivo. While exploring the potential of claudin-4 overexpression as a therapeutic intervention to enhance barrier function we found that neither knockout nor overexpression of claudin-4 affected tight junction permeability. Claudin-4 was, however, able to enhance barrier function when expressed along with claudin-2. These and other preliminary data indicate that, contrary to conventional wisdom, claudin-4 does not form barriers, but rather reduces permeability by directly disrupting claudin-2 polymers. Our patch-clamp studies showing that tight junction channels are actively gated suggest that less extreme approaches to modifying permeability, such as stabilizing the closed state of the channel, are possible. More nuanced approaches could be therapeutically important, as our in vivo studies indicate that complete claudin-2 channel inhibition can be detrimental. For example, loss of claudin-2-mediated paracellular water and Na+ efflux resulted in defective pathogen clearance by claudin-2 knockout mice. Separate studies of occludin led to our unexpected observation that, by mechanisms unrelated to barrier function, occludin is a key regulator of epithelial survival. In parallel we discovered that ZO-1 is required for epithelial orientation, proliferation, and apical structure. These data make it clear that studying these proteins individually will lead to incomplete understanding and will limit utility of the findings. This proposal therefore seeks to understand the relationships between diverse tight junction protein functions, the molecular interactions that direct these activities, and the potential of that knowledge to guide development of structure-based, rational therapies.